Synthesis, crystal structure, redox behavior and comprehensive studies on DNA binding and cleavage properties of transition metal complexes of a fluoro substituted thiosemicarbazone derived from ethyl pyruvate

Polyhedron 34 (2012) 149–156

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Synthesis, crystal structure, redox behavior and comprehensive studies on DNA binding and cleavage properties of transition metal complexes of a fluoro substituted thiosemicarbazone derived from ethyl pyruvate Aishakhanam H. Pathan a, Raghavendra P. Bakale a, Ganesh N. Naik a, Christopher S. Frampton b, Kalagouda B. Gudasi a,⇑ a b

Department of Chemistry, Karnatak University, Dharwad 580 003, Karnataka, India SAFC Pharmorphix, A Sigma–Aldrich Company, 250 Cambridge Science Park, Milton Road, Cambridge CB4 0WE, UK

a r t i c l e

i n f o

Article history: Received 9 November 2011 Accepted 21 December 2011 Available online 10 January 2012 Keywords: Ethyl pyruvate DNA binding Escherichia coli DNA Crystal structure

a b s t r a c t Air and moisture stable coordination compounds of late first row transition metals, viz. Mn(II), Co(II), Ni(II), Cu(II) and Zn(II), with a newly designed ligand, ethyl 2-(2-(4-fluorophenylcarbamothioyl)hydrazono)propanoate (LH), were prepared and fully characterized using various spectro-analytical techniques. The title compound acts as a tritopic, monobasic chelating ligand with S, N and O as the donor sites and is preferably found in the thiol form in all the complexes studied. The single crystal X-ray structure of the ligand (LH) was determined at 100 K and it was shown to be monoclinic, space group C2/c, with two molecules of LH and one molecule of water in the asymmetric unit giving an overall hemi-hydrate stoichiometry. All the metal complexes were found to be octahedral in nature with a 1:2 metal–ligand stoichiometry except for [Cu(L)ClH2O] which has a square pyramidal geometry with a 1:1 metal–ligand ratio. A quasi reversible cyclic voltammogram was obtained for the Cu(II) complex and was assigned to the Cu(II)/Cu(III) couple, whereas Ni(II) exhibited an irreversible anodic peak. Binding and cleavage properties of all the compounds to Escherichia coli DNA were comprehensively investigated by electronic absorption spectroscopy, viscosity, electrochemistry and gel electrophoresis measurements. They are found to be competent binding agents, offering coupled products at room temperature after incubation. Moreover the results of agarose gel electrophoreses showed that [Co(L)2] and [Ni(L)2] in the series have high binding and cleavage affinity towards DNA. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The synthesis of compounds incorporating thiosemicarbazones has attracted widespread attention of chemists as well as biologists, mainly due to their diverse biological activity in pharmaceutical and agrochemical fields [1–4]. The biological properties of thiosemicarbazones are often related to metal ion coordination, for instance the lipophilicity, which is related to the tendency of a molecule to be transported through biological membranes, can be modified by coordination, and further the metal in the complex has its own influence in enhancing the overall activity of the complex compared to the free ligand through a synergic effect [5,6]. Design of small model molecules that can mimic natural biomolecules are of considerable importance because of their potential application in medicine [7–9]. Knowledge of the interactions of these external small molecules with DNA is important to understand how natural biomolecules function in biological systems. ⇑ Corresponding author. Tel.: +91 836 2215286; fax: +91 836 2771275. E-mail address: [email protected] (K.B. Gudasi). 0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2011.12.033

Among these small molecules, transition metal complexes containing multidentate ligands have gained much attention due to their unique coordination properties and significant physicochemical properties and also their possible application as new therapeutic agents. The ligands in the metal complexes play a major role in their binding to DNA. Large planar ligands promote intercalative binding of the metal complexes to DNA [10,11], whereas the central metal ion plays a crucial role in the cleavage of DNA. Prodrugs of ethyl pyruvate and its derivatives have potential antitumour effects and transition metal complexes having carboxylate ligands with additional donor atoms, such as nitrogen and sulfur, are revealed as new structural types which may lead to compounds with potential activity [12–14]. Therefore a thiosemicarbazone derived from ethyl pyruvate could be a molecule with potential activity. It could also reveal valuable information in understanding the mode of binding to DNA for the design of new probes of helical conformations and also anti-tumor drugs. As an attempt to understand the relationship between the structures of thiosemicarbazone, its transition metal complexes and their intercalating mode to DNA, here we report the complete

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characterization of the thiosemicarbazone ligand, including its single crystal X-ray structure, its transition metal complexes and their binding and cleavage properties to Escherichia coli DNA.

2. Experimental 2.1. Materials and physical measurements All the reagents were commercially available (Aldrich or Merck) and were used as supplied. Solvents were purified and dried according to standard procedures. The thiosemicarbazone was prepared according to earlier reports, with slight modifications [15–17]. The metal chlorides used were in the hydrated form. The metal content of the complexes was determined according to standard methods [18]. 1H and 13C NMR spectra were recorded on a BRUKER 500 MHz spectrometer in DMSO-d6 with tetramethylsilane (TMS) as an internal standard. The molar conductivity was measured on an ELICO-CM-82 conductivity bridge. Infrared spectra of the ligand and its metal complexes were recorded in KBr discs in the region 4000–400 cm1 on a Nicolet 170 SX FT-IR spectrometer. Magnetic measurements were made on a Johnson Matthey magnetic susceptibility balance at room temperature using Hg[Co(CNS)4] as the calibrant. All the compounds were analyzed for carbon, hydrogen, nitrogen and sulfur using a Thermo quest elemental analyzer. The UV–Vis spectra of all the compounds in DMSO were recorded on a Varian Cary 50 Bio UV–Vis spectrophotometer. Thermal analysis of the metal complexes were carried out on a Universal V2.4F TA instrument, keeping the final temperature at 1000 °C and the heating rate maintained at 10 °C/min. The cyclic voltammetric experiments were carried out with a three electrode apparatus using a CHI1110A electrochemical analyzer (USA). Electron paramagnetic resonance (EPR) spectra of the Cu(II) complex were recorded at room temperature and LNT on a Varian E-4 X-band spectrometer using tetracyanoethylene (TCNE) as the g-marker. The Ostwald viscometer was used for hydrodynamic measurements.

2.2. Preparation of ligand: ethyl 2-(2-(4-fluorophenylcarbamothioyl) hydrazono)propanoate [LH] The ligand was prepared in two steps. First, 4-(4-fluorophenyl)thiosemicarbazide was obtained and purified by the literature method [19]. In the second step, ethyl pyruvate (0.116 g, 1 mmol) was added dropwise to a 20 ml methanolic solution of 4-(4-fluorophenyl)thiosemicarbazide (0.185 g, 1 mmol) and heated under reflux for 4 h, then allowed to cool to ambient temperature. The yellow crystalline solid that formed was filtered, washed and recrystallised from methanol (Scheme 1). Yellow colored crystals, suitable for single crystal X-ray diffraction analysis, were obtained by slow evaporation of its methanolic solution. Yield: 89%; M.p: 105–108 °C. 1 H NMR (DMSO-d6) d: 11.09 (s, 1H, hydrazine NH), 9.99 (s, 1H, NH phenyl), 4.21 (m, 2H, CH2), 2.17 (s, 3H, N@C–CH3), 1.26 (t, 3H, CH3); 13 C NMR (DMSO-d6) d: 178.04 (HN–C@S), 164.16 (COOEt), 139.85 (C@N), 61.23 (CH2), 13.09 (CH3). Anal. Calc. for C12H14FN3O2S: C, 50.87; H, 4.98; N, 14.83; S, 11.32. Found: C, 50.85; H, 4.98; N, 14.7;

H N F

H N

S, 11.30%. IR (cm1): m(C@O) 1703; m(NH) phenyl 3320; m(NH) hydrazine 3181; m(C@N) 1652; m(C–O) 1246. kmax (nm): 260, 276, 308, 332. 2.2.1. Crystal structure analysis of ethyl 2-(2-(4-fluorophenylcarbamo thioyl)hydrazono)propanoate [LH] The selected crystal of LH was mounted on the tip of a 200 lm Mitagen loop with perfluorinated oil and cooled rapidly to 100 K in a stream of cold nitrogen. Data were collected on an Oxford Diffraction (Agilent Technologies) SuperNova X-ray diffractometer equipped with an Oxford Cryosystems Cobra low temperature device using Cu Ka radiation (k = 154.178 Å) from a SuperNova Cu X-ray micro source and focusing mirror optics. The structures were solved by direct methods and refined against F2 by full-matrix least-squares using the program SHELXTL [20]. The positional coordinates of the heteroatom hydrogen atoms were all located from the Fourier difference map and freely refined along with an isotropic temperature parameter. The remaining hydrogen atoms were positioned geometrically and refined using a riding model (including free rotation about the methyl C  C bond), with C  H = 95–99 Å and with Uiso(H) = 1.2 (1.5 for methyl groups) times Ueq of the parent carbon atom. Full data collection and refinement details are given in Table 1. 2.3. Preparation of metal complexes A mixture of the transition metal chloride, viz. Mn(II), Co(II), Ni(II), Cu(II) and Zn(II), (1 mmol) and the ligand [LH] (0.283 g, 1 mmol) in methanol was stirred at room temperature for 4 h. The precipitate obtained was washed with cold methanol and dried in vacuo. Attempts to grow single crystal of the complexes were unsuccessful. 2.3.1. [Zn(C12H13FN3O2S)2]H2O 1 H NMR (DMSO-d6) d ppm: 10.49 (s, 1H, NH phenyl), 4.20 (m, 2H, CH2), 2.16 (s, 3H, N@C–CH3), 1.26 (t, 3H, CH3); 13C NMR (DMSO-d6) d ppm: 164.16 (COOEt), 162.33 (New@N–C@N), 139.85 (C@N), 61.23 (CH2), 13.09 (CH3). Anal. Calc. for [Zn(C12H13FN3O2S)2]H2O: C, 44.48; H, 4.36; N, 12.97; S, 9.90; Zn, 10.09. Found: C, 44.48; H, 4.35; N, 12.98; S, 9.93; Zn, 10.05%. IR (cm1): m(C@O) 1657; m(NH) phenyl 3290; m(C@N) 1604; m(C–S) 709; m(C–O) 1254. kmax (nm): 281, 316, 367, 460. Molar conductivity (ohm1 cm2 mol1): 1.56. 2.3.2. [Mn(C12H13FN3O2S)2] leff: 5.91 BM. Anal. Calc. for [Mn(C12H13FN3O2S)2]: C, 46.53; H, 4.23; N, 13.56; S, 10.35; Mn, 8.87. Found: C, 46.55; H, 4.21; N, 13.55; S, 10.35; Mn, 8.90%. IR (cm1): m(C@O) 1657; m(NH) phenyl 3322; m(C@N) 1592; m(C–S) 715; m(C–O) 1255. kmax (nm): 333, 348, 401, 469, 674. Molar conductivity (ohm1 cm2 mol1): 3.07. 2.3.3. [Co(C12H13FN3O2S)2] leff: 4.11 BM. Anal. Calc. for [Co(C12H13FN3O2S)2]: C, 46.23; H, 4.20; N, 13.40; S, 10.28; Co, 9.44. Found: C, 46.26; H, 4.18; N, 13.42; S, 10.29; Co, 9.43%. IR (cm1): m(C@O) 1649; m(NH) phenyl 3253; m(C@N) 1618; m(C–S) 742; m(C–O) 1249. kmax (nm): 290, 388, 455, 687. Molar conductivity (ohm1 cm2 mol1): 3.54.

O NH2

S

O

+ O

Thiosemicarbazide

H N

Reflux for 4hrs Methanol

F

Ethyl pyruvate Scheme 1. Synthetic route for the ligand (LH).

H N S

O

N O

Thiosemicarbazone [LH]

A.H. Pathan et al. / Polyhedron 34 (2012) 149–156 Table 1 Crystallographic data for the ligand LH. Crystal data Empirical formula Formula mass Crystal size (mm) Crystal system Space group a (Å) b (Å) c (Å) b (°) V [Å3  106] Z Dcalc (g cm3) l (cm1) (Mo Ka) T (K) 2hmax (°) Index range h k l Reflections collected Unique reflections (Rint) Reflections with Fo > 4r(Fo) Number of parameters R1 wR2 (all data) Maximum and minimum residual density ((e Å3)  106)

C12H15FN3O2.50S 292.33 0.40  0.35  0.35 monoclinic C2/c 2507.02(5) 1310.73(3) 1665.69(4) 100.017(2) 5390.1(2) 16 1.441 0.259 100 52.74 31 ? 30 13 ? 16 20 ? 20 16 262 5506 (0.0242) 4848 380 0.0307 0.0800 0.315, 0.254

were fitted to the above equation and graph was obtained with a slope equal to 1/(eb  ef) and intercept equal to 1/[K(eb  ef)]. Hence Kb was obtained from the ratio of the intercept to the slope [21]. Viscosity measurements were carried out using an Oswald micro-viscometer maintained at a constant temperature (26.0 ± 0.1 °C) in a thermostat. The DNA concentration was kept constant in all samples (100 lM), but the complex concentration was increased each time (from 50 to 200 lM). Mixing of the solution was achieved by bubbling nitrogen gas through the viscometer. The flow time was measured with a digital stop watch. The sample flow times were measured three times and the mean value was considered for calculation. Data are presented in a plot of (g/g0)1/3 versus the ratio [complex]/[DNA], where g and g0 are the specific viscosity of DNA in presence and in absence of the complex, respectively. The values of g and g0 were calculated by using the equation:

g ¼ ðt  t0 Þ=t0

2.3.4. [Ni(C12H13FN3O2S)2] leff: 3.12 BM. Anal. Calc. for [Ni(C12H13FN3O2S)2]: C, 46.25; H, 4.20; N, 13.40; S, 10.29; Ni, 9.42. Found: C, 46.27; H, 4.18; N, 13.37; S, 10.26; Ni, 9.46%. IR (cm1): m(C@O) 1652; m(NH) phenyl 3274; m(C@N) 1606; m(C–S) 670; m(C–O) 123. kmax (nm): 276, 298, 372, 617, 913. Molar conductivity (ohm1 cm2 mol1): 2.23. 2.3.5. [Cu(C12H13FN3O2S)ClH2O] leff: 1.86 BM. Anal. Calc. for [Cu(C12H13FN3O2S)ClH2O]: C, 36.09; H, 3.79; N, 10.52; S, 8.03; Cu, 16.67; Cl, 8.88. Found: C, 36.13; H, 3.75; N, 10.57; S, 8.01; Cu, 16.63; Cl, 8.74%. IR (cm1): m(C@O) 1658; m(NH) phenyl 3235; m(C@N) 1619; m(C–S) 717; m(C–O) 1261. kmax (nm): 311, 364, 464, 720. Molar conductivity (ohm1 cm2 mol1): 20.56. 2.4. Biochemistry 2.4.1. DNA interaction studies The concentration of DNA per nucleotide [C(p)] was measured by using its known extinction coefficient at 260 nm (6600 M1 cm1). The absorbance at 260 nm (A260) and at 280 nm (A280) for DNA was measured to check its purity. The ratio A260/A280 was found to be 1.78, indicating that DNA was satisfactorily free from protein. Tris buffer [5 mM tris (hydroxymethyl)aminomethane, pH 7.2, 50 mM NaCl] was used for the absorption, viscosity and electrochemistry experiments. The spectroscopic titrations were carried out by adding increasing amounts of DNA to a solution of the complex at a fixed concentration contained in a quartz cell. The UV–Vis spectra were recorded after equilibration at 26.0 ± 0.1 °C for 10 min after each addition. The intrinsic binding constant Kb was determined from the plot of [DNA]/(ea  ef) versus [DNA], according to Eq. (1).

½DNA=ðea  ef Þ ¼ ½DNA=ðeb  ef Þ þ 1=½K b ðeb  ef Þ

151

ð1Þ

where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients ea, ef and eb correspond to Aobs/[complex], extinction coefficient for the free complex and the extinction coefficient of the complex in the totally bound form, respectively. The data

where ‘t’ is the observed flow time of a solution containing DNA and the complexes and ‘t0’ is the flow time of the DNA solution alone. The relative viscosities for DNA were calculated from the relation (g/g0) [22]. The cyclic voltammetric experiments were carried out with a three electrode apparatus using a CHI1110A electrochemical analyzer (USA). The complexes were dissolved in DMSO to the desired concentrations. Cyclic voltammograms of fixed concentrations of the complexes in the absence and presence of DNA were measured. The shifts in the values of DEp (separation of the anodic and cathodic peak potentials), ipc/ipa (the ratio of the cathodic to anodic peak currents), and the formal potential E1/2 explains the binding ability of the complexes with DNA. 2.5. Nuclease activity using gel electrophoreses For the gel electrophoresis experiments [23], solutions of the complexes in DMSO (1 mg/ml) were prepared and these test samples (1 lg) were added to the genomic DNA samples of E. coli and incubated for 2 h at 37 °C. Agarose gel was prepared in TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA/l pH 7.3), the solidified gel obtained at 55 °C was placed in an electrophoresis chamber flooded with TAE buffer. Then 20 ll each of the incubated complex–DNA mixtures (mixed with bromophenol blue dye at a 1:1 ratio) was loaded on the gel along with the standard DNA marker, and the electrophoresis was carried out under the TAE buffer system at 50 V for 2 h. At the end of the electrophoresis, the gel was carefully stained with EtBr (ethidium bromide) solution (10 lg/ml) for 10–15 min and visualized under UV light using a Bio-Rad Trans illuminator. The illuminated gel was photographed by using a Polaroid camera (a red filter and Polaroid film were used). 3. Results and discussion Analytical and spectroscopic data for the ligand [LH] and its metal complexes indicate a 1:1 metal–ligand stoichiometry for [Cu(L)ClH2O] and 1:2 for the [Mn(L)2], [Co(L)2], [Ni(L)2] and [Zn(L)2](H2O) complexes. The proposed structures of the complexes are given in Scheme 2. These complexes are soluble in solvents like DMF and DMSO. The molar conductance values (1.56–20.5 X1 cm2 mol1) suggest a non-electrolytic nature of complexes in DMSO at a concentration of 103 M [24]. 3.1. Infrared spectral studies The two medium intensity bands in the region 3270–3140 cm1 in the spectrum of the ligand are assigned to v(NH) phenyl and

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exhibits relatively the same resonances as observed in the 1H NMR spectrum of the ligand.

v(NH) hydrazine. The v(NH) stretching frequency at 3140 cm1 is absent in the spectra of all the complexes, indicating the deprotonation of the hydrazine NH and subsequent coordination through the thiolate functionality [25]. This fact is further supported by considerable reduction in intensity of the thioamide bands (around 1350 and 950 cm1) which were assigned to the coupled vibration of v(C@S). A considerable low shift (45–50 cm1) of v(C@O) in all the complexes compared to the corresponding ligand was observed, indicating its involvement in coordination to the metal ion. The band arising from the >C@N stretching of the free ligand, observed at 1652 cm1, also underwent a change in frequency and intensity, caused by complexation. This band shifts to a lower frequency by 30–40 cm1 in all the complexes [26]. In conclusion, these data suggest a SNO tetradentate behavior of the ligand.

3.3. Mass and EPR spectral studies The mass spectrum of the ligand (Supplementary Fig. S2) shows a molecular ion peak m/z at 283 (M+) and this corresponds to the molecular weight of the ligand. The EPR spectrum of the Cu(II) complex is recorded at both room temperature (RT – 300 K) and liquid nitrogen temperature (LNT), and both spectra exhibit a signal, giving giso = 2.09 and 2.07 for RT and LNT, respectively, with no hyperfine splitting, and hence the complex is assumed to be isotropic in nature [27]. 3.4. Electronic spectral and magnetic studies

3.2. 1H and

13

The electronic spectral assignments and effective magnetic moment values are summarized in Section 2. The representative spectra are shown in Supplementary Fig. S3(a–c). The electronic spectrum of the ligand show bands around 276, 308 and 332 nm. The first band, 276 nm, is assigned to a ligand p ? p⁄ transition, while the band around 308 nm is assigned to an n ? p⁄ transition associated with the imine function of the thiosemicarbazone. Another band observed in the 332 nm region is assigned to an n ? p⁄ transition originating from the thioamide function of the thiosemicarbazone [28]. The nickel complex exhibits a magnetic moment of 3.12 BM, which is compatible with an octahedral Ni(II) environment [29]. The electronic spectrum of this complex generally has three spinallowed transitions in Oh symmetry. The lowest energy band in 3 the 913 nm region was assigned to a [3T2g A2g](m1) transition, while the other bands in the regions 617 nm and 370–390 nm 3 3 may be assigned to m2[(F) 3T1g A2g ] and m3[3T2g(P) A2g(P)] transitions, respectively. These observations support an octahedral geometry for the complex [30]. The Cu(II) complex possesses a magnetic moment of 1.86 BM, corresponding to the spin-only value for a square pyramidal complex. The absorption spectrum of this complex exhibits kmax values at 311, 364 and 464 nm. The first two peaks can be assigned to p ? p⁄ and n ? p⁄ transitions of the ligands. The third band around 464 nm is assigned to a ligand to metal S ? Cu(II) charge transfer transition. The d–d transition was observed as a broad band around 720 nm, suggesting a square pyramidal geometry for the complex [31]. The Co(II) complex possesses a magnetic moment of 4.11 BM, which is in agreement with the spin only value for an octahedral

C NMR spectral studies

The 1H, 13C and H-HCOSY NMR spectra of the ligand and the zinc complex were recorded in DMSO-d6-solvent on a BRUKER 500 MHz instrument. Representative spectra are given in Supplementary Fig. S1(a–d). The two singlets observed in the 1H NMR spectrum of the ligand at 11.05 and 9.99 ppm are due to hydrazine NH and phenyl NH protons, respectively. The peak due to the hydrazine NH proton was absent, while the peak due to the phenyl NH proton is observed down field in the spectrum of the Zn(II) complex. This observation clearly supports the involvement of the >C@S chromophore in coordination via thioenolization. The aromatic ring protons, observed as multiplets in the region 7.19–7.58 ppm in the spectrum of ligand, are shifted slightly downfield in the spectrum of the Zn(II) complex. A multiplet at 4.21 ppm and a triplet at 1.26 ppm are assigned to >CH2 and –CH3 protons of the ester group, respectively. The N@C–CH3 protons are observed as a singlet at 2.17 ppm in the ligand and at 2.16 ppm in the zinc complex. In the 13C NMR spectrum of the ligand the signal at 178.04 ppm corresponds to the thioamide carbon (HN–C@S), which disappears in the 13C NMR spectrum of the Zn(II) complex, and a new signal at 162.33 ppm (assigned to the new >C@N) indicates the coordination of sulfur via deprotonation. The peaks at 164.16 and 139.85 ppm are assigned to >C@N and >C@O, respectively. The methylene and methyl carbon signals of the ester group are observed at 61.23 and 13.99 ppm, respectively. The 13C NMR spectrum of the Zn(II) complex suggests coordination through the carbonyl oxygen, azomethine nitrogen and thioamide sulfur atoms. The assignments of the proton signals were also obtained from 2D COSY NMR, which

H N

N S

F

H N

O

N O

O O

N

N

O .H2O

Zn

F

S

O

N

S

F

M

N

O N H

O

N

M= Co, Ni, Mn H N F

N

O

N

S

O Cu

H2O

F

S

Cl

Scheme 2. Proposed structures of the complexes.

N

N H

A.H. Pathan et al. / Polyhedron 34 (2012) 149–156

153

Co(II) environment. The broad peak observed around 370–390 nm is assigned to a LMCT transition. The peaks around 450–460 and 640–670 nm were attributed to 4T1g(F) ? 4T1g(P) and 4T1g ? 4A2g transitions, suggesting an octahedral structure for the complex [30]. The electronic spectrum of the Mn(II) complex shows bands around 280–300, that were assigned to the n ? p⁄ transition of the carbonyl group, whereas the charge transfer transitions are observed around 350–390 nm. The d–d transition bands are not observed as a very weak tail of the ligand absorption into the visible region is enough to mask the d–d bands. The magnetic moment of Mn(II) complex is found to be 5.91 BM. The zinc complex is diamagnetic with a d10 configuration, and hence does not show any d–d transitions. 3.5. Single crystal X-ray structure of LH The single crystal X-ray structure of the ligand, LH, was determined at 100 K and was shown to be monoclinic, space group C2/c, with two molecules of LH and one molecule of water in the asymmetric unit, giving an overall hemi-hydrate stoichiometry (Fig. 1). The bond lengths and angles are as expected for a substituted thiosemicarbazone and are given in Table 2 [15]. The water molecule forms four strong intermolecular hydrogen bonds with the two independent molecules in the asymmetric unit to form a discrete 2:1 hemi-hydrate cluster. The intermolecular hydrogen bonds comprise of two donor interactions (O1W–HW1B  O1A, [284.1(2) Å, 165(2)°] and O1W–HW1A  O1B, [284.5(2) Å, 164(2)°]) and two acceptor interactions (N1A–H1AA  O1W, [292.5(2) Å, 160(2)°] and N1B–H1BA  O1W, [299.3(2) Å, 160(2)°]). The two fluorophenyl groups of the cluster are oriented to form a strong edge to face aromatic interaction (Supplementary Fig. S4), with closest contact distances, C2B  H2AA, 290.5 Å and C6B  H3AA, 313.4 Å. 3.6. Cyclic voltammetric studies The cyclic voltammetric experiment was performed at room temperature in DMSO under O2 free conditions in the potential range 1.0 to +1.0 V, using a glassy carbon working electrode (0.082 cm2), a platinum counter electrode and an Ag/Ag+ reference electrode. The ligand and the complexes (except the Cu(II) and Ni(II) complexes) did not show any electrochemical response over the working potential range. The voltammograms of both the Cu(II) and Ni(II) complexes (Supplementary Fig. 5(a–b)) showed a single electron transfer process. For the Cu(II) complex, an oxidation peak was observed in the range 0.120–0.250 V during the anodic potential scan and a reduction peak in the range 0.120–0.023 V was observed during the cathodic scan. The separation between the anodic and cathodic peak potentials DEp = Epa  Epc was found to be 230 mV, and the ratio of cathodic to anodic peak currents, ipc/ ipa was 1. This suggests a quasi-reversible redox process assignable to the Cu(II)/Cu(III) couple, as evidenced by the following criteria: (i) the peak-to-peak separation DEp is greater than 59 mV, (ii) the current ratios ipc/ipa are constantly nearly equal to 1, and E1/2 = [Epc + Epa/2] is found to be 0.185 V [32]. The voltammogram of the Ni(II) complex shows only one irreversible oxidation peak in the region 0.067 to 0.010 V, assignable to Ni(II)/ Ni(III). For an irreversible oxidation process, the number of electrons transferred per molecule (n) can be calculated from the following equation [33].

jEpa  Epa=2 j ¼ 1:857RT=ð1  aÞnF

ð2Þ

where Epa is the potential of the oxidation peak, Epa/2 is the half peak potential of the oxidation peak, a is the electron transfer coefficient (generally, 0.3 < a < 0.7), F is the Faraday constant (96 487 C mol1), R is the universal gas constant (8.314 J K1 mol1) and T is the

Fig. 1. Molecular structure of the ligand (LH). Thermal ellipsoids are drawn at the 50% probability level. Hydrogen atoms are displayed with an arbitrarily small radius. Hydrogen bonds between the ligand and the water molecule are shown as solid dashed lines and the aromatic edge to face interaction as thin dashed lines.

temperature in Kelvin. From the voltammogram of the Ni(II) complex, the value of |Epa  Epa/2| was determined to be 0.1005 V, so according to Eq. (2), the number of electrons transferred was calculated to be 1.18 when ‘a’ was assumed to be 0.6, which suggests the involvement of one electron per molecule. 3.7. Thermal studies The thermal stability and decomposition pattern of the complexes were analyzed by TG and DTA studies. Representative thermograms are given in Supplementary Fig. S6(a–b). It is observed that the decomposition of the Cu(II) complex takes place in two stages. The first step corresponds to a mass loss of 4.82% in the range 130–170 °C, attributed to the loss of one coordinated water molecule. The corresponding DTA peak at 161 °C in the spectrum signifies it is an endothermic process. In the second stage, the decomposition corresponds to a mass loss of 89.11% in the range 220–450 °C, showing the combined loss of one chloride and the ligand, with the respective DTA curve at 258 °C representing an endothermic process. For the decomposition of the Zn(II) complex, the mass loss of 3.11% in the range 95–110 °C is attributed to the loss of one lattice held water molecule and the mass loss of 89% around 200–450 °C corresponds to the decomposition of the ligand. The final decomposition products were analyzed to be the respective metal oxides. Thermograms of the Co(II) and Ni(II) complexes show no weight loss in the region 30–200 °C, indicating the absence of coordinated or lattice held solvent molecules. Weight losses of 87.4% and 86.22%, in the temperature range 200–400 °C, are due to the loss of the ligand, respectively. The plateau obtained after heating the complexes above 700 °C corresponds to the formation of the respective stable metal oxides. 3.8. DNA binding/cleavage studies 3.8.1. Absorption studies The binding of complexes and drugs to the DNA helix has been characterized classically through absorption spectral titrations, by following the changes in absorbance and the shift in the wavelength as a function of the added concentration of DNA. In the

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Table 2 Selected bond distances and bond angles of LH. Atoms

Bond distance (Å)

Atoms

Bond distance (Å)

Atoms

Bond angles (°)

Atoms

Bond angles (°)

F1A–C4A O1A–C10A O2A–C10A S1A–C7A O2A–C11A N1A–C7A N2A–N3A N2A–C7A N3A–C8A

1.3613 1.2071 1.3308 1.6660 1.4611 1.3377 1.3539 1.3803 1.2847

F1B–C4B O1B–C10B O2B–C10B S1B–C7B O2B–C11B N1B–C7B N2B–N3B N2B–C7B N3B–C8B

1.3644 1.2126 1.3249 1.6738 1.4628 1.3359 1.3646 1.3778 1.2851

C10A–O2A–C11A C7A–N1A–C1A N3A–N2A–C7A C6A–C1A–N1A N1A–C7A–N2A N1A–C7A–S1A N3A–C8A–C10A O1A–C10A–C8A O2A–C10A–C8A

116.48 125.37 120.31 121.15 114.83 127.03 112.91 124.80 111.24

C10B–O2B–C11B C7B–N1B–C1B N3B–N2B–C7B C6B–C1B–N1B N1B–C7B–N2B N1B–C7B–S1B N3B–C8B–C10B O1B–C10B–C8B O2B–C10B–C8B

116.53 128.00 120.94 123.65 114.80 128.22 113.23 124.55 111.02

3.8.2. Viscosity measurements As a means for further clarifying the binding nature of complexes with DNA, the viscosity of DNA solutions containing varying amounts of added complexes were measured. In the absence of crystallographic structure data, hydrodynamic measurements are regarded as one of the least ambiguous and the most critical tests of molecular binding in solution [22]. A classical intercalative molecular interaction causes a significant increase in viscosity of the DNA solution due to the increase in separation of the base pairs at the intercalation sites and hence an increase in the overall DNA length. The values of (g/g0)1/3 [where g and g0 are the specific viscosity of DNA in the presence and absence of the complex, respectively] are plotted against [compound]/[DNA] (Supplementary Fig. S8). The results reveal that the presence of the complexes increases the relative viscosity of the DNA solutions. From the extent of the increased relative viscosity of the DNA solution, an intercalative interaction can be ruled out. The change in viscosity caused by the nickel and cobalt complexes is more than the others in the series, which indicates a strong intercalating mode of interaction of these complexes. 3.8.3. Cyclic voltammetry studies Cyclic voltammetry is also a useful technique for investigating the interaction of metal complexes with DNA [35,36]. In this paper the DNA-binding ability of electrochemically active species, i.e. the Cu(II) and Ni(II) complexes, were investigated by cyclic voltammetry. The nickel complex in the absence of DNA showed an oxidation peak in the region 0.067 to 0.010 V [chosen as an analytical signal in the following studies in the scan range after being electrolyzed at 0.4 V for 60 s]. After interaction with 100 lL DNA, it was found

2.5

[DNA]/(Ea-Ef) 10-9

present investigation it has been used to monitor the interaction of the ligand and its metal complexes (500 lM) with E. coli DNA, in different concentrations (10–50 lM). In the case of the nickel complex (as a representative example) the broad peak at 372 nm was monitored as a function of added DNA (Fig. 2). The observations reveal that increasing the amount of DNA results in a decrease in the molar absorptivity (hypochromism) as well as a blue shift by 8–10 nm (hypsochromic shift). This indicates an intercalative mode of interaction of the complex with DNA. The intrinsic binding constant Kb was determined by the plot of [DNA]/(ea  ef) versus [DNA]. The value of Kb for the nickel complex is 3.31  104 M1 and suggests a moderate intercalative interaction when compared with the classical intercalators (Kb 106–107) [34]. The cobalt complex shows hypochromism with a hypsochromic shift of 25–30 nm for the peak at 687 nm (d–d transition) upon addition of increasing amounts of DNA, indicating an intercalative binding mode. The binding constant, Kb = 1.10  105 M1, suggests a strong interaction between DNA and the complex (Supplementary Fig. S7(a–b)). The binding constants of 3.71  103, 4.08  103 and 2.41  103 calculated for the copper, zinc and manganese complexes, respectively, suggest a weaker interaction.

Kb= 1.10 X 105

2.0 1.5 1.0 0.5 10

20

30

40

50

[DNA] 10-6

Fig. 2. Absorption spectrum of the nickel complex in the absence and presence of increasing amounts of E. coli DNA. Inset: plot of [DNA]/(ea  ef) vs. [DNA].

that the oxidation peak (Supplementary Fig. S9) showed a positive shift in the peak potential Ep by 0.013 V, with a decrease in the peak current by 0.147, suggesting that the complex has interacted with DNA. The decreasing extent of the peak current observed for the complex upon addition of DNA indicates that the complex interacts with DNA in an intercalating way. In the absence of DNA, the electrochemical behavior of the Cu(II) complex shows a redox process (Fig. 3, curve ‘a’), corresponding to the Cu(II)/Cu(III) couple. On the addition of DNA, the complex experiences a shift in E1/2 by 0.023 V as well as in Ep values, as shown in curve ‘b’. The ratio of ipc/ipa for the bound complex is less than unity, suggesting that DNA is bound to the complex. In addition to the changes in the formal potential, the voltammetric peak decreases upon addition of DNA to the complex. The decrease in the current is due to the diffusion of the equilibrium mixture of free and DNA-bound metal complex to the electrode surface [37]. These data suggest that both complexes bind to DNA. The higher decrease of the peak currents observed for the Ni(II) complex compared to the Cu(II) complex upon addition of DNA suggest that the binding affinity of the former to DNA is stronger than that of the later.

3.8.4. DNA cleavage study by gel electrophoresis Suitably designed metal complexes, after binding to DNA, can induce several changes in the DNA conformation, such as bending, ‘local denaturation’ (overwinding and underwinding), intercalation, micro loop formation and subsequent DNA shortening, leading to a decrease in the molecular weight of the DNA. The photograph, as shown in Fig. 4, shows bands with different bandwidths compared to the control and marker, and this is the differentiating criteria for binding and cleavage abilities of the complexes with E. coli DNA in this study. A control experiment using DNA alone does not show any significant cleavage of DNA, even after a long exposure time (Lane 1). Further, when genomic

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to the Cu(II)/Cu(III) couple, whereas the Ni(II) complex exhibits one irreversible anodic peak. The DNA-binding properties of all the complexes were comprehensively studied by different methods, including electronic absorption spectroscopy, viscosity, cyclic voltammetry and gel electrophoresis methods. The experimental results showed that the ligand and all the complexes could intercalatively bind to DNA with a medium intrinsic association strength. The Co(II) and Ni(II) complexes show strong binding and a cleavage affinity towards the DNA of E. coli, which is further confirmed by the gel electrophoreses study. Acknowledgments

Fig. 3. Cyclic voltammograms of the copper complex in the absence (a) and presence (b) of 100 lM DNA in Tris–HCl of pH 7.4, scan rate = 150 mV s1.

The authors thank USIC, Karnatak University, Dharwad for providing the spectral facilities. Recording of ESR spectra from IIT Bombay is gratefully acknowledged. One of the authors (Aishakhanam H. Pathan) is thankful to UGC for providing financial assistance under its Research Fellowship in Science for Meritorious Students. Appendix A. Supplementary data CCDC 831784 contains the supplementary crystallographic data for compound LH. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@ccdc. cam.ac.uk. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.poly.2011.12.033. References

Fig. 4. Agarose gel electrophoresis pattern for the cleavage of E. coli DNA with the ligand and the complexes.

DNA is allowed to interact with the complexes, a substantial decrease in the intensities of the bands (Lane LH, Cu, Zn, Mn) for the metal bound DNA as compared to the untreated control DNA was observed, which suggests an intercalative binding mode for the complexes. In the case of the nickel and cobalt complexes (Lanes 5 and 6), the bands completely disappeared, indicating the sufficient cleavage of DNA. This cleavage may be because of the intercalation of the pyruvate units with the DNA strands induces enzyme-mediated DNA cleavage. The results suggest that sufficient binding of the ligand and all the metal complexes (except the cobalt and nickel complexes) causes a change in the conformation of genomic DNA, whereas the cobalt and nickel complexes have shown potential chemical nuclease activity.

4. Conclusions The new thiosemicarbazone analog (LH) of ethyl pyruvate was designed and synthesized in good yield. The 1H and 13C NMR data and single crystal X-ray diffraction were successfully used to elucidate the formation of the Schiff base. The single crystal X-ray structure demonstrated the material in its crystalline form from methanol exists as a hemi-hydrate, with the formation of a 2:1 ligand:water hydrogen bonded cluster. The transition metal complexes of the ligand were synthesized and characterized using various physico-chemical analyses. The ligand acts as a tridentate monobasic chelate with SNO as the donating sites. UV and magnetic moment data supports a square pyramidal geometry for the copper complex and an octahedral geometry for the cobalt, nickel, manganese and zinc complexes. A quasi-reversible cyclic voltammogram is obtained for the Cu(II) complex, which was assigned

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